United States Patent
`
`[19]
`5,829,448
`[11] Patent Number:
`[45] Date of Patent: Nov. 3, 1998
`Fisher et al.
`
`
`
`U8005829448A
`
`[54] METHOD FOR IMPROVED SELECTIVITY
`IN PHOTO-ACTIVATION OF MOLECULAR
`AGENTS
`
`[75]
`
`Inventors: Walter G. Fisher, Knoxville; Eric A.
`Wachter, Oak Ridge; H. Craig Dees,
`Knoxville, all of Tenn.
`
`[73] Assignee: Photogen, Inc., Knoxville, Tenn.
`
`[21] Appl. No.: 739,801
`
`[22]
`
`Filed:
`
`Oct. 30, 1996
`
`Int. Cl.6 ..................................................... A61B 00/19
`[51]
`
`.. 128/898; 604/20
`[52] US. Cl.
`............
`[58] Field of Search ............................... 128/898; 607/89,
`607/2, 3; 604/20
`
`[56]
`
`References Cited
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`8/1993 Santana—Blank ..
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`
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`606/9
`12/1996 Hu ............................................... 606/9
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`(List continued on next page.)
`
`Primary Examiner—V. Millin
`Assistant Examiner—Kelly O’Hara
`Attorney, Agent, or Firm—Richard M. Kessler
`
`[57]
`
`ABSTRACT
`
`A method for the treatment of a particular volume of plant
`or animal tissue comprising the steps of treating the plant or
`animal tissue with at least one photo-active molecular agent,
`wherein the particular volume of the plant or animal tissue
`retains at least a portion of the at least one photo-active
`molecular agent, and then treating the particular volume of
`the plant or animal tissue with light sufficient to promote a
`simultaneous two-photon excitation of at least one of the at
`least one photo-active molecular agent retained in the par-
`ticular volume of the plant or animal tissue, wherein the at
`least one photo-active molecular agent becomes active in the
`particular volume of the plant or animal tissue. There is also
`disclosed a method for the treatment of cancer in plant or
`animal
`tissue and a method for producing at
`least one
`photo-activated molecular agent in a particular volume of a
`material.
`
`68 Claims, 23 Drawing Sheets
`
`14
`
`Vibrational
`
`Second
`Absorbed
`Photon
`
`Photon \-"\
`Photon
`
`
`
`
`wt
`Absorbed
`
`1
`Excited
`State
`
`26 /\_/
`Virtual
`.
`... . .-
`Level k
`
`First
`Absorbed
`Photonv\
`
`Ground
`State
`
`/
`2
`
`First
`Absorbed
`Photon\’\
`
`52Winternal
`Conversion
`
`\Virtual
`Level
`
`
`
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 1
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 1
`
`

`

`5,829,448
`
`Page 2
`
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`Alcon Research, Ltd.
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`
`Alcon Research, Ltd.
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`
`

`

`5,829,448
`Page 3
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 3
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 3
`
`

`

`US. Patent
`
`Nov. 3, 1998
`
`Sheet 1 0f 23
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`5,829,448
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 4
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 4
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`US. Patent
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 13
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 13
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`

`

`US. Patent
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`Nov. 3, 1998
`
`Sheet 11 0123
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`5,829,448
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`Figure11
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 14
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 14
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`

`

`
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 15
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`

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`US. Patent
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`Nov. 3, 1998
`
`Sheet 13 0f 23
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`5,829,448
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`Figure 13
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 16
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 16
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`

`

`US. Patent
`
`Nov. 3, 1998
`
`Sheet 14 0f 23
`
`5,829,448
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 17
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 17
`
`

`

`US. Patent
`
`Nov. 3, 1998
`
`Sheet 15 0f 23
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`5,829,448
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`164
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 18
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` Figure15
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 18
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`

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`US. Patent
`
`Nov. 3, 1998
`
`Sheet 16 0f 23
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`5,829,448
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`
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`
`156
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 19
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 19
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`

`

`US. Patent
`
`Nov. 3, 1998
`
`Sheet 17 0f 23
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 20
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 20
`
`

`

`US. Patent
`
`Nov. 3, 1998
`
`Sheet 18 0f 23
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`5,829,448
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 21
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 21
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`

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`US. Patent
`
`Nov. 3, 1998
`
`Sheet 19 0f 23
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`5,829,448
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`Exhibit 1023 - Page 22
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`Alcon Research, Ltd.
`Exhibit 1023 - Page 22
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`Nov. 3, 1998
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`Nov. 3, 1998
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`Exhibit 1023 - Page 26
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`

`

`5,829,448
`
`1
`METHOD FOR IMPROVED SELECTIVITY
`IN PHOTO-ACTIVATION OF MOLECULAR
`AGENTS
`
`This invention was made with Government support
`under Contract No. DE-AC05-84OR21400 awarded by the
`US. Department of Energy to Lockheed Martin Energy
`Systems, Inc. Lockheed Martin Energy Systems, Inc., and
`the Oak Ridge Associated Universities have waived rights to
`this invention to the inventors. The Government has rights in
`this invention pursuant
`to Contract No. DE-AC05-
`840R21400 awarded by the US. Department of Energy.
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to methods and
`apparatus for achieving selective photo-activation of one or
`more molecular agents with a high degree of spatial control.
`The method taught for achieving selective photo-activation
`utilizes the special properties of non-linear optical energy
`for exciting or promoting an agent from one molecular
`energy level to another with a high degree of spatial and
`molecular specificity. The special features of this method are
`applicable in the processing of various types of materials,
`and in particular afford distinct advantages in the treatment
`of diseases in humans and animals. Specifically, use of
`non-linear excitation methods facilitate controlled therapeu-
`tic activation of photodynamic therapy agents in deep tissue
`using near infrared to infrared radiation, which is absorbed
`and scattered to a lesser extent than methods and radiations
`
`currently used.
`
`BACKGROUND OF THE INVENTION
`
`An urgent need exists in many fields for a method that is
`capable of selectively controlling the activation of various
`molecular agents. The desired improvements in activation
`include enhancements in spatial or temporal control over the
`location and depth of activation, reduction in undesirable
`activation of other co-located or proximal molecular agents
`or structures, and increased preference in the activation of
`desirable molecular agents over that of undesirable molecu-
`lar agents. Various linear and non-linear photo-chemical and
`photo-physical methods have been developed to provide
`some such improvements for some such agents. However, in
`general the performance and applicability of these methods
`have been less than desired. Specifically, improved photo-
`activation methods are needed that may be used to selec-
`tively photo-activate a variety of molecular therapeutic
`agents while providing improved performance in the control
`of application of this photo-activation.
`Application of optical radiation for probing or transfor-
`mation of molecular agents has been known for many years.
`Linear optical excitation has been extensively studied as a
`means for achieving semi-selective activation of molecular
`therapeutic agents. For example, Tessman et al.
`(J. W.
`Tessman, S. T. Isaacs and J. E. Hearst, “Photochemistry of
`the Furan-Side 8-Methoxypsoralen-Thymidine Monoadduct
`Inside the DNA Helix. Conversion to Diadduct and to
`
`Pyrone-Side Monoadduct,” Biochemistry, 24 (1985)
`1669—1676)
`teach of the application of light at specific
`energies as a means for achieving partial selectivity in the
`formation of molecular bonds between target molecular
`agents and DNA (deoxyribonucleic acid). Kennedy et al. (J.
`C. Kennedy, R. H. Pottier and D. C. Ross, “Photodynamic
`Therapy with Endogenous Protoporphyrin IX: Basic Prin-
`ciples and Present Clinical Experience,” Journal of Photo-
`chemistry and Photobiology, B: Biology, 6 (1990) 143—148)
`
`10
`
`15
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`20
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`25
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`review progress on development and application of various
`photosensitive molecular agents for clinical treatment of
`disease. And Teuchner et al. (K. Teuchner, A. Pfarrherr, H.
`Stiel, W. Freyer and D. Leupold, “Spectroscopic Properties
`of Potential Sensitizers for New Photodynamic Therapy
`Start Mechanisms via Two-Step Excited Electronic States,”
`Photochemistry and Photobiology, 57 (1993) 465—471)
`teach of the use of spectroscopic properties for selection of
`candidate photo-active agents. Yet performance of these
`agents and specifically the methods used for their activation
`have not been as successful as desired. For example, Young
`(A. R. Young, “Photocarcinogenicity of Psoralens Used in
`PUVA Treatment: Present Status in Mouse and Man,” Jour-
`nal of Photochemistry and Photobiology, B: Biology, 6
`(1990) 237—247) presents strong evidence that the optical
`radiation used in common treatment regimes based on linear
`optical excitation of photosensitive molecular agents can
`itself produce disease and other undesirable side effects.
`Furthermore, a less than desirable penetration depth has
`plagued most efforts at linear optical excitation of molecular
`therapeutic agents, primarily as a consequence of the effects
`of optical scatter and of absorbance of the incident probe
`radiation at wavelengths near the linear absorption bands of
`these agents. In fact, virtually all examples of the use of
`linear optical excitation for molecular transformation are
`plagued by fundamental performance limits that are attrib-
`utable to undesirable absorption and scatter of the incident
`optical radiation by the surrounding matrix, poor specificity
`in excitation of probe molecular species, and a lack of
`suitable physical mechanisms for precise control of the
`extent and depth of activation.
`Various non-linear optical excitation methods have been
`employed in an effort to achieve specific improvements in
`the selectivity of photo-activation for certain applications,
`and to address many of the limitations posed by linear
`excitation methods. Excitation sources ranging from single-
`mode, continuous wave (CW) lasers to pulsed Q-switched
`lasers having peak powers in excess of 1 GW have been
`employed with these methods. For example, Wirth and Lytle
`(M. J. Wirth and F. E. Lytle, “Two-Photon Excited Molecu-
`lar Fluorescence in Optically Dense Media,” Analytical
`Chemistry, 49 (1977) 2054—2057) teach use of non-linear
`optical excitation as a means for stimulating target mol-
`ecules present
`in optically dense media;
`this method is
`shown to be useful in limiting undesirable direct interaction
`of the probe radiation with the media itself, and provides a
`means for effectively exciting target molecular agents
`present in strongly absorbing or scattering matrices. Yeung
`et al. teach further use of non-linear optical excitation for
`highly specific excitation of target molecules present in very
`small volumes (M. J. Sepaniak and E. S. Yeung, “Laser
`Two-Photon Excited Fluorescence Detection for High Pres-
`sure Liquid Chromatography,” Analytical Chemistry, 49
`(1977) 1554—1556; M. J. Sepaniak and E. S. Yeung, “High-
`Performance Liquid Chromatographic Studies of Coal Liq-
`uids by Laser-Based Detectors,” Journal of
`Chromatography, 211 (1981), 95—102; and W. D. Pfeffer and
`E. S. Yeung, “Laser Two-Photon Excited Fluorescence
`Detector for Microbore Liquid Chromatography,” Analyti-
`cal Chemistry, 58 (1986) 2103—2105). These works teach of
`the attractive performance advantages of non-linear optical
`excitation of target molecular agents present in complex
`matrices, specifically where reduced background excitation,
`low probe volumes, and complementary selection rules
`provided by non-linear methods aid in increasing selectivity
`of the analysis. Improved spatial control over the active
`region has been further developed by Wirth (M. J. Wirth and
`
`Alcon Research, Ltd.
`Exhibit 1023 - Page 27
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`Alcon Research, Ltd.
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`5,829,448
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`3
`H. O. Fatunmbi, “Very High Detectability in Two-Photon
`Spectroscopy,” Analytical Chemistry, 62 (1990) 973—976);
`specifically, Wirth teaches a method for achieving extremely
`high spatial selectivity in the excitation of target molecular
`agents using a microscopic imaging system. Similar control
`has been further applied by Denk et al. (W. Denk, J. P.
`Strickler and W. W. Webb, “Two-Photon Laser Microscopy,”
`US. Pat. No. 5,034,613) who teach of a special confocal
`laser scanning microscope utilizing non-linear laser excita-
`tion to achieve intrinsically high three-dimensional control
`in the photo-activation of various molecular fiuorophor
`agents on a cellular or sub-cellular scale. This microscope is
`used to excite molecular fiuorophor agents added to biologi-
`cal specimens, which constitute an optically dense medium;
`the special properties of non-linear optical excitation are
`utilized to substantially limit excitation to a confocal region
`occurring at the focus of an objective lens, thereby allowing
`the possibility of three-dimensional
`imaging by sharply
`controlling the depth of focus. Control of photo-excitation
`for generation of luminescence-based images at the cellular
`and subcellular level is shown in target samples mounted on
`a stage. This microscope is also used for localized photolytic
`release of caged effector molecules present in individual
`cells mounted on a stage, and is claimed to be useful for
`inducing additional photochemical reactions in such cells.
`However,
`reduction in photo-induced necrosis of cells
`located at the focal plane is claimed to be the primary benefit
`of this microscopy approach, based on the replacement of
`ultraviolet excitation radiation with near infrared radiation.
`
`While the substantial body of prior art exemplified by
`these cited examples clearly demonstrates many attractive
`features of photo-activation methods, a general method for
`achieving selective photo-activation of one or more molecu-
`lar agents with a high degree of spatial control that is capable
`of meeting the diverse needs of industry has not been
`previously taught. Specifically, practical methods for effect-
`ing such control on scales that are significant for therapeutic
`uses or for general materials processing applications have
`not been previously taught.
`invention to
`Therefore,
`it
`is an object of the present
`provide a method for the treatment of plant or animal tissue
`with a high degree of spacial selectivity.
`It is further object of the present invention to provide such
`a method using a light source and photo-active materials to
`enhance the high degree of spacial selectivity.
`It is another object of the present invention to provide
`such a method using wavelengths of light which are less
`harmful to the plant or animal tissue than the wavelengths of
`light currently used for the treatment of plant or animal
`tissue.
`
`It is yet another object of the present invention to provide
`such a method using light which is less prone to scatter in
`and absorption by plant or animal tissue than the wave-
`lengths of light currently used for the treatment of plant or
`animal tissue.
`
`Consideration of the specification, including the several
`figures and examples to follow, will enable one skilled in the
`art to determine additional objects and advantages of the
`invention.
`
`SUMMARY OF THE INVENTION
`
`Having regard to the above and other objects and
`advantages, the present invention generally provides for a
`method for the treatment of a particular volume of plant or
`animal tissue comprising the steps of treating the plant or
`animal tissue with at least one photo-active molecular agent,
`
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`4
`wherein the particular volume of the plant or animal tissue
`retains at least a portion of the at least one photo-active
`molecular agent, and then treating the particular volume of
`the plant or animal tissue with light sufficient to promote a
`simultaneous two-photon excitation of at least one of the at
`least one photo-active molecular agent retained in the par-
`ticular volume of the plant or animal tissue, wherein the at
`least one photo-active molecular agent becomes active in the
`particular volume of the plant or animal tissue.
`The present invention also provides for a method for the
`treatment of cancer in plant or animal tissue comprising the
`steps of treating the plant or animal tissue with at least one
`photo-active molecular agent, wherein the cancer in the
`plant or animal tissue retains at least a portion of at least one
`of the at least one photo-active molecular agent, and treating
`the plant or animal tissue with light sufficient to promote a
`simultaneous two-photon excitation of the at
`least one
`photo-active molecular agent retained in the cancer in the
`plant or animal tissue, wherein the at least one photo-active
`molecular agent becomes active in the cancer in the plant or
`animal tissue.
`
`The present invention further provides for a method for
`producing at least one photo-activated molecular agent in a
`particular volume of a material. The method comprises
`treating the particular volume of the material with light
`sufficient to promote a simultaneous two-photon excitation
`of at least one photo-active molecular agent contained in the
`particular volume of the material. The at least one photo-
`active molecular agent
`then becomes a photo-activated
`molecular agent in the particular volume of the material. In
`preferred embodiments of the present invention the material
`is selected from the group consisting of plant tissue and
`animal tissue and the material is pretreated with at least one
`photo-active molecular agent such that the material retains at
`least a portion of the photo-active agent at the time that the
`particular volume of the material is treated with light suf-
`ficient to promote a simultaneous two-photon excitation of
`the photo-active molecular agent.
`The present invention also provides for a method for
`producing at least one photo-activated molecular agent in a
`particular volume of a material comprising treating the
`particular volume of the material with light sufficient to
`promote optical excitation of at
`least one photo-active
`molecular agent contained in the particular volume of the
`material, wherein the at least one photo-active molecular
`agent becomes a photo-activated molecular agent
`in the
`particular volume of the material.
`In an additional preferred embodiment of the present
`invention,
`the light sufficient
`to promote a simultaneous
`two-photon excitation of the p